AIM-Ia
We have developed a new strategy for activity-based probes to address the members of the proteome that target N-acyllysine posttranslational modifications, which are highly prevalent and constitute a continuously expanding collection of chemical groups. Our results are highly encouraging, as they corroborate the hypothesis that we can install enzymatically stable amide bond analogues, while retaining the ability of native interaction partners in cell lysates to recognize the modification with high selectivity and specificity. In addition, we provide the first investigation of the importance of peptide sequence on performance of N-acyllysine-containing photo cross-linking probes (Bæk et al., Chem. Eur. J. 2019).
The developed methodology provides basis for detailed chemo proteomics studies by combining our protocol with tandem mass spectrometry-based methods.
AIM-Ib
With the enhanced structural integrity of the probes, we expect that potential reader domains can be identified as well. These efforts are initiated and are still ongoing beyond the timeframe of the Action, through a collaboration with the laboratory of Prof. Luca Laraia at the Technical University of Denmark.
Our work on erasers of epsilon-N-lactyllysine and epsilon-N-(beta-hydroxybutyryl)lysine, performed in collaboration with the laboratory of Prof. YingMing Zhao at the University of Chicago and published this year (Moreno-Yruela et al., Science Advances 2022), served as inspiration for the latest series of probes.
AIM-II
Development of potent and selective SIRT5 inhibitor:
Early in the Action we succeeded in publishing the most potent and selective inhibitors of SIRT5 enzyme activity (Rajabi et al. Angewandte Chemie 2017). This is a landmark achievement in inhibition of SIRT5, which could be of therapeutic interest. Furthermore, the developed compound has now been commercialized by a company specialized in assay materials and tool compounds, which makes the molecule available to the scientific community beyond the labs that have already requested samples from us.
We then embarked on two different strategies to improve cell uptake. Both our prodrug strategy and bioisostere strategy produced potent compounds that exhibited target engagement in living cells and were able kill SIRT5-dependent cancer cells (Rajabi, Hansen, et al., Angewandte Chemie 2022).
Finally, arylfluorosulfates have recently emerged as latent electrophiles (reviewed by us; Martin-Gago et al. Angewandte Chemie 2019). We have thus incorporated arylfluorosulfates into tour scaffold and demonstrated SIRT5 labeling in cells (Bolding et al.,Angewandte Chemie 2022).
Insights into mechanism-based inhibitors of SIRT1–3:
We have evaluated a series of mechanism-based inhibitors of SIRT1–3. Notably, we have shown that short-chain thioamide-based inhibitors can be efficiently deacylated by SIRT1–3 to an unprecedented degree (Rajabi, Nielsen, et al., ACS Med. Chem. Lett. 2020).
The collective insight gained in this study is essential for future sirtuin inhibitor design and underscores the crucial need for comprehensive compound assessment.
Development of potent and selective SIRT2 inhibitors:
We have built on previous knowledge to develop the most potent SIRT2 inhibitors reported to date, which exhibit inhibition of cell migration and invasion in breast cancer cells in culture (Nielsen et al., RSC Chem. Biol. 2021).
Development of potent and selective SIRT3 inhibitor:
To achieve selectivity for SIRT3 over SIRT1 and SIRT2, a mitochondrial targeting sequence was incorporated into the inhibitors. Thus, achieving selectivity by sub-cellular localization rather than selective affinity for the target enzyme alone (Troelsen et al., RSC Chem. Biol. 2021).
